A 4,103 marker integrated physical and comparative map of the horse genome

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.     

Construction of a 5000<Subscript>rad</Subscript> whole-genome radiation hybrid panel in the horse and generation of a comprehensive and comparative map for ECA11

A 5000_rad whole-genome radiation hybrid (RH) panel was created for the horse. The usefulness of the panel for generating physically ordered maps of individual equine chromosomes was tested by typing 24 markers on horse Chromosome 11 (ECA11). The overall retention of markers on this chromosome was 43.6%. Almost complete retention of two of the typed markers—CA062 and AHT44—clearly indicated the location of thymidine kinase gene on the short arm of ECA11. Seven of the typed markers were FISH mapped to align the RH and cytogenetic maps. With the RH-MAPPER approach, a physically ordered map comprising four linkage groups and incorporating all the markers was obtained. The study provides the first comprehensive map for a horse chromosome that integrates all available mapping data and adds new information that spans the entire length of the equine chromosome. The map clearly underlines the resolving power and utility of the panel and emphasizes the need to have uniformly distributed cytogenetic markers for appropriate alignment of RH map with the chromosome. A comparative status of the ECA11 map in relation to the corresponding human/mouse chromosome is presented. Received: 7 June 2001 / Accepted: 4 October 2001     

High-resolution RH map of horse chromosome 22 reveals a putative ancestral vertebrate chromosome

High-resolution gene maps of individual equine chromosomes are essential to identify genes governing traits of economic importance in the horse. In pursuit of this goal we herein report the generation of a dense map of horse chromosome 22 (ECA22) comprising 83 markers, of which 52 represent specific genes and 31 are microsatellites. The map spans 831 cR over an estimated 64 Mb of physical length of the chromosome, thus providing markers at approximately 770 kb or 10 cR intervals. Overall, the resolution of the map is to date the densest in the horse and is the highest for any of the domesticated animal species for which annotated sequence data are not yet available. Comparative analysis showed that ECA22 shares remarkable conservation of gene order along the entire length of dog chromosome 24, something not yet found for an autosome in evolutionarily diverged species. Comparison with human, mouse, and rat homologues shows that ECA22 can be traced as two conserved linkage blocks, each related to individual arms of the human homologue-HSA20. Extending the comparison to the chicken genome showed that one of the ECA22 blocks that corresponds to HSA20q shares synteny conservation with chicken chromosome 20, suggesting the segment to be ancestral in mammals and birds.     

Conservation of gene order between horse and human X chromosomes as evidenced through radiation hybrid mapping

A radiation hybrid (RH) map of the equine X chromosome (ECAX) was obtained using the recently produced 5000(rad) horse x hamster hybrid panel. The map comprises 34 markers (16 genes and 18 microsatellites) and spans a total of 676 cR(5000), covering almost the entire length of ECAX. Cytogenetic alignment of the RH map was improved by fluorescent in situ hybridization mapping of six of the markers. The map integrates and refines the currently available genetic linkage, syntenic, and cytogenetic maps, and adds new loci. Comparison of the physical location of the 16 genes mapped in this study with the human genome reveals similarity in the order of the genes along the entire length of the two X chromosomes. This degree of gene order conservation across evolutionarily distantly related species has up to now been reported only between human and cat. The ECAX RH map provides a framework for the generation of a high-density map for this chromosome. The map will serve as an important tool for positional cloning of X-linked diseases/conditions in the horse.     

High-resolution gene maps of horse chromosomes 14 and 21: additional insights into evolution and rearrangements of HSA5 homologs in mammals

High-resolution physically ordered gene maps for equine homologs of human chromosome 5 (HSA5), viz., horse chromosomes 14 and 21 (ECA14 and ECA21), were generated by adding 179 new loci (131 gene-specific and 48 microsatellites) to the existing maps of the two chromosomes. The loci were mapped primarily by genotyping on a 5000-rad horse x hamster radiation hybrid panel, of which 28 were mapped by fluorescence in situ hybridization. The approximately fivefold increase in the number of mapped markers on the two chromosomes improves the average resolution of the map to 1 marker/0.9 Mb. The improved resolution is vital for rapid chromosomal localization of traits of interest on these chromosomes and for facilitating candidate gene searches. The comparative gene mapping data on ECA14 and ECA21 finely align the chromosomes to sequence/gene maps of a range of evolutionarily distantly related species. It also demonstrates that compared to ECA14, the ECA21 segment corresponding to HSA5 is a more conserved region because of preserved gene order in a larger number of and more diverse species. Further, comparison of ECA14 and the distal three-quarters region of ECA21 with corresponding chromosomal segments in 50 species belonging to 11 mammalian orders provides a broad overview of the evolution of these segments in individual orders from the putative ancestral chromosomal configuration. Of particular interest is the identification and precise demarcation of equid/Perissodactyl-specific features that for the first time clearly distinguish the origins of ECA14 and ECA21 from similar-looking status in the Cetartiodactyls.     

A radiation hybrid map of bovine chromosome 24 and comparative mapping with human chromosome 18

We present herein a bovine chromosome 24 (BTA24) radiation hybrid (RH) map using 40 markers scored on a panel of 90 RHs. Of these markers, 29 loci were ordered with odds of at least 1000:1 in a framework map. An average retention frequency of 17.4% was observed, with relatively higher frequencies near the centromere. The length of the comprehensive map was 640 centiray5000 (cR5000) with an average marker interval of approximately 17.3 cR5000. The observed locus order is generally consistent with currently published bovine linkage and physical maps. Nineteen markers were either Type I loci or closely associated with expressed sequences and thus could be used to compare the BTA24 RH map with human mapping information. All genes located on BTA24 were located on human chromosome 18, and previously reported regions of conserved synteny were extended. The comparative data revealed the presence of at least six conserved regions between these chromosomes.     

A high-resolution physical map of equine homologs of HSA19 shows divergent evolution compared with other mammals

A high-resolution (1 marker/700 kb) physically ordered radiation hybrid (RH) and comparative map of 122 loci on equine homologs of human Chromosome 19 (HSA19) shows a variant evolution of these segments in equids/Perissodactyls compared with other mammals. The segments include parts of both the long and the short arm of horse Chromosome 7 (ECA7), the proximal part of ECA21, and the entire short arm of ECA10. The map includes 93 new markers, of which 89 (64 gene-specific and 25 microsatellite) were genotyped on a 5000-rad horse × hamster RH panel, and 4 were mapped exclusively by FISH. The orientation and alignment of the map was strengthened by 21 new FISH localizations, of which 15 represent genes. The approximately sevenfold-improved map resolution attained in this study will prove extremely useful for candidate gene discovery in the targeted equine chromosomal regions. The highlight of the comparative map is the fine definition of homology between the four equine chromosomal segments and corresponding HSA19 regions specified by physical coordinates (bp) in the human genome sequence. Of particular interest are the regions on ECA7 and ECA21 that correspond to the short arm of HSA19—a genomic rearrangement discovered to date only in equids/Perissodactyls as evidenced through comparative Zoo-FISH analysis of the evolution ofancestral HSA19 segments in eight mammalian orders involving about 50 species.     

A radiation hybrid comparative map of ovine chromosome 1 aligned to the virtual sheep genome

Ovis aries chromosome one (OAR1) is the largest submetacentric chromosome in the sheep genome and is homologous to regions on human chromosomes 1, 2, 3 and 21. Using the USUoRH5000 ovine whole-genome radiation hybrid (RH) panel, we have constructed a RH map of OAR1 comprising 102 framework and 75 placed/binned markers across five linkage groups spanning 3759.43 cR5000, with an average marker density of 21.2 cR5000/marker. The alignment of our OAR1 RH map shows good concordance with the recently developed virtual sheep genome, with fewer than 1.86% discrepancies. A comparative map of OAR1 was constructed by examining the location of RH-mapped orthologues in sheep within the genomes of cow, human, horse and dog. Analysis of the comparative map indicates that conserved syntenies within the five ovine RH linkage groups underwent internal chromosomal rearrangements which, in general, reflect the evolutionary distances between sheep and each of these four species. The ovine RH map presented here integrates all available mapping data and includes new genomic information for ovine chromosome 1.     

A high-resolution comparative radiation hybrid map of ovine chromosomal regions that are homologous to human chromosome 6 (HSA6)

In this study, we constructed high-resolution radiation hybrid (RH) and comparative maps of ovine chromosomes or chromosomal segments that are homologous to human chromosome 6 (HSA6). A total of 251 markers were successfully genotyped across the recently developed USUoRH5000 whole-genome panel; 208 of these markers were assigned to five RH linkage groups distributed on three ovine chromosomes (OAR8, 9 and 20). The RH maps have good correspondence with previous chromosome painting data, although a small centromeric region on OAR9 that is homologous to HSA6 had not been previously detected using human chromosome paints on ovine chromosomal spreads. High percentages of the ovine markers were identified as orthologues in the bovine (86.3%), dog (85.8%), horse (69.3%) and human (88.7%) genomes. These maps contribute to investigations in mammalian chromosome evolution and the search for economic trait loci in sheep.     

A radiation hybrid map of sheep chromosome 23 based on ovine BAC-end sequences

More than 375,000 BAC-end sequences (BES) of the CHORI-243 ovine BAC library have been deposited in public databases. blastn searches with these BES against HSA18 revealed 1806 unique and significant hits. We used blastn-anchored BES for an in silico prediction of gene content and chromosome assignment of comparatively mapped ovine BAC clones. Ovine BES were selected at approximately 1.3-Mb intervals of HSA18 and incorporated into a human-sheep comparative map. An ovine 5000-rad whole-genome radiation hybrid panel (USUoRH5000) was typed with 70 markers, all of which mapped to OAR23. The resulting OAR23 RH map included 43 markers derived from BES with high and unique BLAST hits to the sequence of the orthologous HSA18, nine EST-derived markers, 16 microsatellite markers taken from the ovine linkage map and two bovine microsatellite markers. Six new microsatellite markers derived from the 43 mapped BES and the two bovine microsatellite markers were linkage-mapped using the International Mapping Flock (IMF). Thirteen additional microsatellite markers were derived from other ovine BES with high and unique BLAST hits to the sequence of the orthologous HSA18 and also positioned on the ovine linkage map but not incorporated into the OAR23 RH map. This resulted in 24 markers in common and in the same order between the RH and linkage maps. Eight of the BES-derived markers were mapped using fluorescent in situ hybridization (FISH), to thereby align the RH and cytogenetic maps. Comparison of the ovine chromosome 23 RH map with the HSA18 map identified and localized three major breakpoints between HSA18 and OAR23. The positions of these breakpoints were equivalent to those previously shown for syntenic BTA24 and HSA18. This study presents evidence for the usefulness of ovine BES when constructing a high-resolution comprehensive map for a single sheep chromosome. The comparative analysis confirms and refines knowledge about chromosomal conservation and rearrangements between sheep, cattle and human. The constructed RH map demonstrates the resolution and utility of the newly constructed ovine RH panel.     

A radiation hybrid map of bovine chromosome one

Radiation hybrid (RH) mapping has proven to be an extremely powerful approach to constructing high density maps of human chromosomes and is experiencing increased use in other animals, including cattle. A 5000 rad bovine whole-genome radiation hybrid panel was recently constructed in order to integrate existing cattle linkage maps with evolutionarily conserved genes and provide high resolution comparative maps relative to humans and mice. We utilized this panel to construct a 19 marker framework map of bovine chromosome 1 (BTA1), which included 8 Type I loci and 11 Type II loci ordered with at least 1000:1 odds. A 35 marker comprehensive map including 15 Type I loci and 20 Type II loci was also produced. Of the 15 Type I loci ordered on the comprehensive map, three are ordered on HSA3 and five are ordered in three blocks on HSA21 on the human cytogenetic maps.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1)

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH(5000)) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.     

A human-horse comparative map based on equine BAC end sequences

In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E 相似文献   

Construction of a medium-density horse gene map

A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH) (using an equine RH(5000) panel) and/or by FISH mapping. A comparison of localizations of 713 genes mapped on the horse genome and on the human genome revealed 59 homologous segments and 131 conserved segments. Two of these homologies (ECA27/HSA8 and ECA12p/HSA11p) had not been previously identified. An enhanced resolution of conserved and rearranged chromosomal segments presented in this study provides clarification of chromosome evolution history.     

A comparative radiation hybrid map of sheep chromosome 10

Comparative radiation hybrid (RH) maps of individual ovine chromosomes are essential to identify genes governing traits of economic importance in sheep, a livestock species for which whole genome sequence data are not yet available. The USUoRH5000 radiation hybrid panel was used to generate a RH map of sheep chromosome 10 (OAR10) with 59 markers that span 1,422 cR over an estimated 92 Mb of the chromosome, thus providing markers every 2 Mb (equivalent to every 24 cR). The markers were derived from 46 BAC end sequences (BESs), a single EST, and 12 microsatellites. Comparative analysis showed that OAR10 shares remarkable conservation of gene order along the entire length of cattle chromosome 12 and that OAR10 contains four major homologous synteny blocks, each related to segments of the homologous human chromosome 13. Extending the comparison to the horse, dog, mouse, and chicken genome showed that these blocks share conserved synteny across species.     

FISH analysis comparing genome organization in the domestic horse (Equus caballus) to that of the Mongolian wild horse (E. przewalskii)

Przewalski's wild horse (E. przewalskii, EPR) has a diploid chromosome number of 2n = 66 while the domestic horse (E. caballus, ECA) has a diploid chromosome number of 2n = 64. Discussions about their phylogenetic relationship and taxonomic classification have hinged on comparisons of their skeletal morphology, protein and mitochondrial DNA similarities, their ability to produce fertile hybrid offspring, and on comparison of their chromosome morphology and banding patterns. Previous studies of GTG-banded karyotypes suggested that the chromosomes of both equids were homologous and the difference in chromosome number was due to a Robertsonian event involving two pairs of acrocentric chromosomes in EPR and one pair of metacentric chromosomes in ECA (ECA5). To determine which EPR chromosomes were homologous to ECA5 and to confirm the predicted chromosome homologies based on GTG banding, we constructed a comparative gene map between ECA and EPR by FISH mapping 46 domestic horse-derived BAC clones containing genes previously mapped to ECA chromosomes. The results indicated that all ECA and EPR chromosomes were homologous as predicted by GTG banding, but provide new information in that the EPR acrocentric chromosomes EPR23 and EPR24 were shown to be homologues of the ECA metacentric chromosome ECA5.     

A 12,000-rad porcine radiation hybrid (IMNpRH2) panel refines the conserved synteny between SSC12 and HSA17

Reverse or bidirectional Zoo-FISH suggests that synteny between porcine chromosome 12 (SSC12) and human chromosome 17 (HSA17) is completely conserved. The construction of a high-resolution radiation hybrid (RH) map for SSC12 provides a unique opportunity to determine whether chromosomal synteny is reflected at the molecular level by comparative gene mapping of SSC12 and HSA17. We report an initial, high-resolution RH map of SSC12 on the 12,000-rad IMNpRH2 panel using CarthaGene software. This map contains a total of 320 markers, including 20 microsatellites and 300 ESTs/genes, covering approximately 4836.9 cR12,000. The markers were ordered in 16 linkage groups at LOD 6.0 using framework markers previously mapped on the IMpRH7000-rad SSC12 and porcine genetic maps. Ten linkage groups ordered more than 10 markers, with the largest containing 101 STSs. The resolution of the current RH map is approximately 15.3 kb/cR on SSC12, a significant improvement over the second-generation EST SSC12 RH7000-rad map of 103 ESTs and 15 framework markers covering approximately 2287.2 cR7000. Compared to HSA17, six distinct segments were identified, revealing macro-rearrangements within the apparently complete synteny between SSC12 and HSA17. Further analysis of the order of 245 genes (ESTs) on HSA17 and SSC12 also revealed several micro-rearrangements within a synteny segment. A high-resolution SSC12 RH12,000-rad map will be useful in fine-mapping QTL and as a scaffold for sequencing this chromosome.     

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.     

Evidence for abundant transcription of non-coding regions in the Saccharomyces cerevisiae genome

Background

The ChickRH6 whole chicken genome radiation hybrid (RH) panel recently produced has already been used to build radiation hybrid maps for several chromosomes, generating comparative maps with the human and mouse genomes and suggesting improvements to the chicken draft sequence assembly. Here we present the construction of a RH map of chicken chromosome 2. Markers from the genetic map were used for alignment to the existing GGA2 (Gallus gallus chromosome 2) linkage group and EST were used to provide valuable comparative mapping information. Finally, all markers from the RH map were localised on the chicken draft sequence assembly to check for eventual discordances.

Results

Eighty eight microsatellite markers, 10 genes and 219 EST were selected from the genetic map or on the basis of available comparative mapping information. Out of these 317 markers, 270 gave reliable amplifications on the radiation hybrid panel and 198 were effectively assigned to GGA2. The final RH map is 2794 cR₆₀₀₀ long and is composed of 86 framework markers distributed in 5 groups. Conservation of synteny was found between GGA2 and eight human chromosomes, with segments of conserved gene order of varying lengths.

Conclusion

We obtained a radiation hybrid map of chicken chromosome 2. Comparison to the human genome indicated that most of the 8 groups of conserved synteny studied underwent internal rearrangements. The alignment of our RH map to the first draft of the chicken genome sequence assembly revealed a good agreement between both sets of data, indicative of a low error rate.